Biology subjects

de Groot, R.

Publications and source records attributed to de Groot, R..

9 recordsLinked to original sources

bioRxiv2025

Allosteric Disordering of eIF2B Regulates the Integrated Stress Response

The ternary complex (TC), composed of translation initiation factor eIF2, GTP, and initiator methionyl tRNA, delivers the first amino acid to the ribosome to initiate protein synthesis. The activity of the decameric eukaryotic initiation factor 2B complex (eIF2B) initiates TC assembly by catalyzing GDP to GTP exchange on eIF2, thereby setting the TC levels in the cell. Stress-induced phosphorylation converts eIF2 from the substrate of the GDP/GTP exchange reaction into an inhibitor (eIF2-P) of eIF2B. This conversion reduces the cells TC levels and induces the widespread reprogramming of translation known as the Integrated Stress Response (ISR). Here, we chart an allosteric axis running through eIF2B, revealing the importance of a protrusive -helix in its {beta}-subunit, the latch-helix, that locks onto the -subunit to induce eIF2B activity. eIF2-P binding unhooks the latch-helix, opening eIF2B, which inhibits its GDP/GTP exchange activity. Distinct viral proteins have convergently evolved to bind to eIF2B and stabilize the latch-helix-bound active state. Using these insights, we generated ISR-ACTivating compounds, ISRACTs, that stabilize eIF2B in its inhibited, unlatched state. Our study thus highlights how state-transitions in eIF2B are regulated via long-range allostery.

biochemistry↗

bioRxiv2025

γδ T cells are the prime anti-tumoral T cells in pediatric neuroblastoma

High-risk pediatric neuroblastoma patients have a dismal survival rate despite intensive treatment regimens. New treatment options are thus required. Even though HLA expression in neuroblastoma is low and immune cell infiltrates are limited, the presence of tumor infiltrating lymphocytes (TILs) is indicative for better patient survival. Here, we show that most tumor lesions contain viable immune cell infiltrates after induction chemotherapy, with high percentages of CD3+ T cells. We therefore expanded the TILs and tested their anti-tumoral activity. With sufficient starting material, TIL expansion was as efficient as for adult solid tumors. However, whereas TIL products from adult tumors almost exclusively contained {beta} T cells, in neuroblastoma-derived TILs, {gamma}{delta} T cells expanded with similar efficacy as {beta} T cells. Importantly, the anti-tumor responses in response to autologous tumor digest primarily originated from (V{delta}1- and V{delta}3-expressing) {gamma}{delta} T cells, and not from {beta} T cells. In conclusion, this finding creates a window of opportunity for immunotherapy for neuroblastoma patients, with {gamma}{delta} T cells as potential prime responders.

cancer biology↗

bioRxiv2024

Tertiary lymphoid structure-related immune infiltrates in NSCLC tumor lesions correlate with low tumor-reactivity of TIL products

Adoptive transfer of tumor infiltrating lymphocytes (TIL therapy) has shown great potential for the treatment of solid cancers, including non-small cell lung cancer (NSCLC). However, not all patients benefit from this therapy, and the parameters that define the likelihood of TIL products to be tumor reactive are to date unknown. Defining prognostic markers that correlate with high level of tumor-reactivity is key for achieving better tailored immunotherapies. To determine whether the composition of immune cell infiltrates correlates with the tumor-reactivity of expanded TIL products, we employed multi-parameter flow cytometry to characterize the immune cell infiltrates from 26 early-stage, and 20 late-stage NSCLC tumor lesions. Unbiased flow cytometry analysis with Cytotree and Spearmans Rank Correlation was used to correlate immune infiltrates with the expansion rate, immune cell activation and T cell differentiation state, and the anti-tumor response of TIL products generated from the same lesions. The composition of tumor immune infiltrates was highly variable between patients, irrespective of the disease stage. High percentages of B cell infiltrates positively correlated with the presence of conventional CD4+ T cells, and an overall increase of naive T cell infiltrates. In contrast, high B cell infiltrates negatively correlated with the tumor-reactivity of expanded TIL products, as defined by cytokine production upon exposure to autologous tumor digest. Tumors with high B cell infiltrates contained IgD+BCL6+ B cells and CXCR5+BLC6+ CD4+ T cell infiltrates and an increased percentage of naive CD8+ T cells, indicative of the presence of tertiary lymphoid structures (TLS) in tumors with high B cell infiltrates. This study reveals that the composition of immune cell infiltrates in NSCLC tumors associates with the functionality of expanded TIL products from NSCLC tumor lesions. Importantly, the tumor-responsiveness of TIL products negatively correlated with the presence of TLS-associated immune infiltrates in tumors. Our finding may thus help improve patient selection for TIL therapy.

immunology↗

bioRxiv2023

Multiscale chromatin dynamics and high entropy in plant iPSC cultures

Plant protoplasts constitute the starting material to induce pluripotent cell masses in vitro competent for tissue regeneration. Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterized by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what are the factors influencing chromatin transitions during culturing is largely unknown. High-throughput imaging and a custom, supervised image analysis protocol extracting over 100 chromatin features unravelled a rapid, multiscale dynamics of chromatin patterns which trajectory strongly depends on nutrients availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating an intrinsic entropy as hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.

molecular biology↗

bioRxiv2022

Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Because current treatment regimens show limited success rates, alternative therapeutic approaches are needed. We recently showed that treatment-naive, stage I/II primary NSCLC tumors contain a high percentage of tumor-reactive T cells, and that these tumor-reactive T cells can be effectively expanded and used for the generation of autologous TIL therapy. Whether these promising findings also hold true for metastatic lesions is unknown yet critical for the translation into the clinic. We here studied the lymphocytic composition and the feasibility to generate tumor-responsive TIL products from metastatic NSCLC lesions that were derived from different locations and from patients who had previously received different treatment regimens, or who were treatment naive. The overall numbers and composition of lymphocyte infiltrates from this various metastatic lesions was by and large comparable to that of early-stage primary NSCLC tumors. With the clinically approved TIL expansion protocol, we effectively expanded TILs from metastatic NSCLC lesions to numbers that were compatible with TIL transfusion, irrespective of the location of the metastasis and of the previous treatment. Importantly, 16 of 21 (76%) tested TIL products displayed anti-tumoral activity, as determined by their production of the key pro-inflammatory cytokines IFN-{gamma}, TNF, IL-2 and of CD137 expression upon incubation with autologous tumor digests by CD8+ and CD4+ T cells. In summary, metastatic NSCLC lesions constitute a viable source for the generation of tumor-reactive TIL products for therapeutic purposes irrespective of their location and the pretreatment regimens.

cancer biology↗

bioRxiv2020

Phase separation induced by cohesin SMC protein complexes

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

biophysics↗

bioRxiv2020

Exosite inhibition of A Disintegrin And Metalloproteinase with Thrombospondin motif (ADAMTS)-5 by a glycoconjugated arylsulfonamide

ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Its aggrecanase activity has been directly linked to the etiology of osteoarthritis (OA), identifying ADAMTS-5 as a pharmaceutical target for OA treatment. However, most existing ADAMTS-5 inhibitors target its active site and therefore suffer from poor selectivity. Here, using a novel approach, we have designed a new class of sugar-based arylsulfonamide inhibitors, which are selective for ADAMTS-5 through binding to a previously unknown substrate-binding site (exosite). Docking calculations combined with molecular dynamics simulations demonstrated that our lead compound is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Targeted mutagenesis identified disintegrin-like domain residues K532 and K533 as an exosite which is critical for substrate recognition. Furthermore, we show that this exosite acts as major determinant for inhibitor binding and, therefore, can be targeted for development of selective ADAMTS-5 inhibitors.

biochemistry↗

bioRxiv2019

AFM images of open and collapsed states of yeast condensin suggest a scrunching model for DNA loop extrusion

Structural Maintenance of Chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex, which compacts DNA during mitosis, was recently shown to be a molecular motor that extrudes large loops of DNA. The mechanism of this unique motor, which takes large steps along DNA at low ATP consumption, remains elusive however. Here, we use Atomic Force Microscopy (AFM) to visualize the structure of yeast condensin and condensin-DNA complexes. Condensin is found to exhibit mainly open O shapes and collapsed B shapes, and it cycles dynamically between these two states over time. Condensin binds double-stranded DNA via a HEAT subunit and, surprisingly, also via the hinge domain. On extruded DNA loops, we observe a single condensin complex at the loop stem, where the neck size of the DNA loop correlates with the width of the condensin complex. Our results suggest that condensin extrudes DNA by a fast cyclic switching of its conformation between O and B shapes, consistent with a scrunching model.

biophysics↗

bioRxiv2018

Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile in situ

Non-small cell lung cancer (NSCLC) is the second most prevalent type of cancer. With the current treatment regimens, the mortality rate remains high. Therefore, better therapeutic approaches are necessary. NSCLCs generally possess many genetic mutations and are well infiltrated by T cells (TIL), making TIL therapy an attractive option. Here we show that T cells from treatment naive, stage I-IVa NSCLC tumors can effectively be isolated and expanded, with similar efficiency as from normal lung tissue. Importantly, 76% (13/17) of tested TIL products isolates from NSCLC lesions exhibited clear reactivity against primary tumor digests, with 0.5%-30% of T cells producing the inflammatory cytokine Interferon (IFN)-{gamma}. Both CD4+ and CD8+ T cells displayed tumor reactivity. The cytokine production correlated well with CD137 and CD40L expression. Furthermore, almost half (7/17) of the TIL products contained polyfunctional T cells that produced Tumor Necrosis Factor (TNF)- and/or IL-2 in addition to IFN-{gamma}, a hallmark of effective immune responses. Tumor-reactivity in the TIL products correlated with high percentages of CD103+CD69+CD8+ T cell infiltrates in the tumor lesions, with PD-1hiCD4+ T cells, and with FoxP3+CD25+CD4+ regulatory T cell infiltrates, suggesting that the composition of T cell infiltrates may predict the level of tumor reactivity. In conclusion, the effective generation of tumor-reactive and polyfunctional TIL products implies that TIL therapy will be a successful treatment regimen for NSCLC patients.

immunology↗